R

Figure 3. Cryptococcus liquefaciens, the yeast species most frequently isolated from subglacial samples

Among the remaining basidiomycetous yeast species detected in lower numbers, Cr. albidus was the most abundant (up to 5 x 105 CFU L-1) [Butinar et al., 2007]. T. mucoides is considered an opportunistic pathogenic species [Kurtzman and Fell, 1998] and it was unexpectedly obtained in high counts (8 x 105 CFU L-1), though only from one glacier [Butinar et al., 2007]. Our group isolated this species also from waters of the Dead Sea [Butinar et al., 2005b]. Surprisingly, half of all the isolated species are repeatedly isolated from humans and only around one quarter of the species (Cr. gilvescens, Cr. macerans, Cr. victoriae, Cystofilobasidium sp., L. fragaria) can be considered psychrophilic, with maximum temperatures for growth not exceeding 25 °C [van Uden, 1984; Vishniac, 1987]. In addition, two not yet described species of the genera Cryptococcus and Cystofilobasidium were discovered in our study [Butinar et al., 2007].

To our knowledge, ascomycetous yeasts have only been exceptionally isolated from cold polar regions [Di Menna, 1966; Atlas et al., 1978; Vishniac, 1987]. This is surprising, as ascomycetous yeasts represent the main spoilage agents of chilled or frozen foods [Davenport, 1980; Schmidt-Lorenz, 1982] and of food preserved with low water activity (aw) [Deak and Beuchat, 1996; Samson et al., 2004]. It is possible that this deficit has been a result of inappropriate selective conditions used to recover the yeasts present in those extreme ecosystems.

In our studies by using low aw media also ascomycetous species D. hansenii and P. guillermondii were frequently found and together with the two dominant basidiomycetous yeasts, Cr. liquefaciens and Rh. mucilaginosa, can probably be considered autochthonous subglacial species. The highest CFU values for D. hansenii were obtained on media with 20% glucose and 5% NaCl (up to 104 CFU L-1), whereas the P. guilliermondii numbers were approximately half (up to 7 x 103 CFU L-1) on the medium with 10% NaCl added, at 24 °C [Butinar et al., sub.]. To our knowledge this counts were the highest ever reported for this two species. However differences in distribution was observed as the two dominant ascomycetous species primarily occurred in Kongsvegen samples (Fig. 4), while the two dominant basidiomycetous species prevailed in samples originating from austre Lovenbreen and Broggerbreen glaciers (Fig. 4) [Butinar et al., 2007, sub.]. In Breggerbreen glacier no ascomycetous yeasts were obtained, probably due to its almost entirely cold base.

D. hansenii, with its anamorphic state Candida famata, is known as a salt- and cold-tolerant species, and it is usually isolated from materials of plant and animal (including clinical) origin, and from soil and air, in temperate climates. The species is ubiquitous in the oceans of the World [Jones, 1976]; however, it can also be isolated from hypersaline water in solar salterns [Butinar et al., 2005b]. It is a common spoilage yeast of frozen food, brine-preserved food and other low aw products [Deak and Beuchat, 1996; Boekhout and Robnet, 2003]. Despite this wide distribution, reports of its occurrence in polar regions are scarce. The presence of D. hansenii has been recorded in Antarctic soils [Atlas et al., 1978], overcooled brine cryopegs in permafrost [Gilichinsky et al., 2005], polar waters [Vishniac, 1987; Ma et al., 1999, 2000; Bridge et al., 2008], and ice from Antarctic glaciers [Di Menna, 1966]. In all cases, the isolates were present at very low densities

P. guilliermondii was the second most frequent ascomycetous yeasts species in the subglacial ice. It was always found in association with D. hansenii, both forming a distinct phylogenetic group [Kurtzman and Robnett, 1998]. P. guilliermondii is also widely distributed in nature, as strains of this species are routinely isolated from exudates of various trees, and from insects [de Araujo et al., 1995; Sibirny, 1996], soil, plants [Capriotti and Ranieri, 1964], the atmosphere and sea water [Diriye et al., 1993], and it was reported as the most frequently occurring species in the Adriatic salterns [Butinar et al., 2005b]. Moreover, P. guilliermondii is among the yeasts that are most commonly related to human disease [Kurtzman and Fell, 1998].

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Figure 4. Principal component analysis of yeast species (1-Candida rugosa-like; 2-Cryptococcus oeirensis, Cr. saitoi, Leucosporidiella fragaria, Trichosporon mucoides; 3-Rhodotorula minuta; 4-Cr. magnus; 5-Rh. laryngis; 6-Cr. albidus; 7-Cr. victoriae, Rhodosporidium diobovatum; 8-Rh. mucilaginosa; 9-Cr. liquefaciens; 10-Cystofilobasidium sp.; 11-Pichia guilliermondii; 12-Filobasidium uniguttulatum; 13-Debaryomyces hansenii; 14-Cr. albidosimilis; 15-Cr. adeliensis; 16-C. parapsilosis, Cr. laurentii; 17-Cr. carnescens) isolated from different samples originating from four glaciers. Letter s, following the name of glacier, indicates subglacial ice with sediment. The first two axes explained 67.3% of the variation in the species data.

Figure 4. Principal component analysis of yeast species (1-Candida rugosa-like; 2-Cryptococcus oeirensis, Cr. saitoi, Leucosporidiella fragaria, Trichosporon mucoides; 3-Rhodotorula minuta; 4-Cr. magnus; 5-Rh. laryngis; 6-Cr. albidus; 7-Cr. victoriae, Rhodosporidium diobovatum; 8-Rh. mucilaginosa; 9-Cr. liquefaciens; 10-Cystofilobasidium sp.; 11-Pichia guilliermondii; 12-Filobasidium uniguttulatum; 13-Debaryomyces hansenii; 14-Cr. albidosimilis; 15-Cr. adeliensis; 16-C. parapsilosis, Cr. laurentii; 17-Cr. carnescens) isolated from different samples originating from four glaciers. Letter s, following the name of glacier, indicates subglacial ice with sediment. The first two axes explained 67.3% of the variation in the species data.

Although it is also known as a food-spoilage yeast of processed and refrigerated food [Diriye et al., 1993], in the polar regions P. guilliermondii has only been reported from permafrost cryopegs [Gilichinsky et al., 2005]. C. parapsilosis, which was isolated from the subglacial ice of a single glacier, is also a food-borne halotolerant yeast [Davenport, 1980; Schmidt-Lorenz, 1982]. Again, it has been frequently isolated from clinical specimens [Kurtzman and Fell, 1998], and it has, somewhat surprisingly, been isolated from solar salterns and from the Dead Sea [Butinar et al., 2005b]. This species has been isolated from the ice tunnel samples, collected at the Amundsen-Scott IGY South Pole Station [Jacobs et al., 1964].

The other ascomycetous yeast species appeared less consistently and with lower counts. C. parapsilosis occurred only in the Conwaybreen glacier, while the C. pseudorugosa-like species was detected only in the Kongsvegen glacier (Table 3) (Fig. 4) [Butinar et al., sub.].

Sampling of ice from a glacier cave resulted in the isolation of a rare species, P. inoueyi (Table 3) [Butinar et al., sub.]. The genus Protomyces may include up to 60 species [Reddy and Kramer, 1975], and as it is poorly studied and so far only six strains are available in culture collections, our finding is of considerable interest for future ecological investigations.

1.4 Penicillium

The cosmopolitan genus Penicillium comprises more than 225, mainly food-, soil-, or airborne species [Pitt et al., 2000]. The genus shows tolerance for cold environments as demonstrated by the fact that many species grow on food preserved in refrigerators [Pitt and Hocking, 1999] or are isolated from alpine, tundra [Domsch et al., 1980], and even polar soil [Frisvad, 2004; McRae et al., 1999]. Being often psychrotolerant and sturdy as well as having prolific production of conidia, it is not surprising that penicillia are among the few viable fungi isolated from glacial ice cores even up to 38,600 years old [Abyzov, 1993]. Spores and fragments of mycelia trapped within ice are exposed to several stressful factors such as freezing, desiccation, and starvation. However, ice also protects microorganisms from UV irradiation, oxidation, and chemical damage [Rogers et al., 2005].

Our study revealed that supraglacial samples contained only up to 50 CFU L-1 of penicillia, whereas subglacial samples harbored a surprisingly rich diversity and high occurrence of penicillia (up to 13x 103 CFU L-1) [Sonjak et al., 2006], equivalent to subglacial bacteria isolated primarily from subglacial debris-rich ice [Skidmore et al., 2000]. It seems that penicillia were present in the soils and sediments that glaciers overrode and became selectively enriched through the processes of melting and freezing that occurred at the glacier bed. The dominant species, both in sediment-rich and in overlying clear glacial ice, is the cosmopolitan P. crustosum [Sonjak et al., 2006], which is typically reported as being food-borne [Samson et al., 2004]. This species was isolated from all sampled glaciers (up to 9,2 x 103 CFU L-1) and represents one of the two dominant species in the glacier outflow water (up to 1,720 CFU L-1) [Sonjak et al., 2006]. It is interesting to note, that P. crustosum strains isolated from the Conwaybreen glacier, differed from all other Arctic strains and also from all tested strains isolated worldwide [Sonjak et al., 2005, 2007a]. Although growth on CREA medium is one of the basic characteristics of this species [Frisvad and Samson, 2004], a group of Arctic isolates showed surprisingly weak growth [Sonjak et al., 2005, 2007a]. Although AFLP analysis distinguished as a new P. crustosum genotype, they did not differ in other tested chemical, physiological, and morphological characteristics [Sonjak et al., 2005, 2007a].

From all isolated penicillia only P. commune, P. discolor, and P. polonicum, were detected with significantly higher counts in clear glacial ice (up to 600 CFU L-1) than in the sediment-rich ice (below 10 CFU L-1) [Sonjak et al., 2006]. These species might have originated in the sediment but they may also have been washed into the subglacial environment from the glacial surface. The only species besides P. crustosum that was detected with high counts in the glacial outflow water was P. bialowiezense (up to 11,3 x 103 CFU L-1) [Sonjak et al., 2006].

A group of Penicillium strains were isolated that did not belong to any known Penicillium species [Sonjak et al., 2007b]. This species was isolated in high numbers from the Kongsvegen subglacial ice and was not detected in the surrounding environment [Sonjak et al., 2007b]. A detailed analysis of secondary metabolite profiles, physiological and morphological characteristics, and partial ß-tubulin gene sequences showed that the proposed new species P. svalbardense is closely related but not identical to P. piscarium and P. simplicissimum [Sonjak et al., 2007b].

Of all the species of Penicillium recovered, most species found in subglacial ice are also among the very common foodborne penicillia, including P. crustosum, P. polonicum, P. discolor, P. commune, P. palitans, P. nordicum, P. solitum, P. echinulatum, P. expansum, P. brevicompactum, P. chrysogenum, and P. tulipae [Sonjak et al., 2006]. In contrast, these species are very rare in soil [Frisvad and Samson, 2004]. The only soilborne forms of Penicillium found in the subglacial ice were P. brasilianum from series Simplisissima, P. lanosum from series Lanosa, P. corylophilum from series Citrina, (all from Penicillium subgenus Furcatum) and P. glabrum and P. thomii from series Glabra, subgenus Aspergilloides [Sonjak et al., 2006]. Thus, penicillia that are primarily foodborne are clearly much more prevalent in subglacial ice than soilborne penicillia. A similar observation was made for penicillia found in the Antarctic, which were isolated from soil or bird nest material on Antarctica. McRae et al. [1999] reported on P. aurantiogriseum, P. brevicompactum, P. chrysogenum, P. commune, P. echinulatum, P. expansum, P. palitans, and P. solitum from subgenus Penicillium, whereas they found P. antarcticum, P. corylophilum, P. fellutanum, P. glabrum, P. janthinellum, P. jensenii, and P. waksmanii of the more soilborne type of penicillia from the subgenera Aspergilloides and Furcatum. This mycobiota is actually rather similar to that found by us in the Arctic subglacial ice. The foodborne penicillia from subgenus Penicillium are extremely rarely found in isolations from soil regardless of climatic zone [Christensen et al., 2000; Frisvad et al., 2000], but apparently they are very common in the polar regions both in soil (Antarctica) and in ice samples (Arctic). Several of the species from subgenus Aspergilloides and Furcatum, including P. glabrum, P. corylophilum, and P. antarcticum, have also been found regularly on foods, whereas some of the fungi reported from Antarctica, such as P. janthinellum, P. jensenii, and P. waksmanii, are common fungi in soil worldwide. These common soilborne types were not found in the subglacial samples. In addition to these species, some new species from soil in Greenland or alpine habitats were also found in the subglacial samples.

Penicillia strains that populate glacial ice must be physiologically adaptable and able to retain their viability throughout the dynamic processes of ice melting and freezing and extremes in pressure. Such enrichment would select for Penicillium populations best adapted to dark, cold, oligotrophic environments with shifting osmotic pressures. These predictions were confirmed by our isolation of the highest Penicillium counts on the enumeration medium used for the isolation of moderate xerophiles at 10°C, whereas almost all penicillia were isolated additionally also on media with even lower water activity. The dominant species, P. crustosum, and the other frequently occurring species apparently can propagate in this extreme habitat due to their sturdy nature, ability to withstand osmotic imbalances, and to adjust to nutritionally poor habitats. Besides, they are characterized by a high production of small-sized conidia, able to spread and colonize pockets and microchannels of the ice. In cold and oligotrophic conditions, small size could be advantageous for more efficient nutrient uptake and for the occupation of microenvironments [Sonjak et al., 2006].

1.5 Aspergillus

The genus Aspergillus and its teleomorphic states contains 254 broadly accepted species [Pitt et al., 2000]. Many of these are known for their xerotolerance and frequent occurrence as contaminants of food preserved with high concentrations of salt or sugar, primarily in warmer climates.

In spite of their ubiquitious nature and preference for low water activity, only few halotolerant Aspergillus species were isolated from Kongsfjorden: A. niger, A. tubigensis, A. sydowii and A. versicolor. A. niger and A. sydowii have been repored world-wide from sand dunes, salt marshes, estuaries, mangrove mud and other marine environments [Gunde-Cimerman et al., 2005a]. Extremotolerant nature of A. sydowii spores enabled it to survive prolonged suspension in Dead Sea water [Kis-Papo et al., 2003], and to survive in the Antarctic ice sheet for 10,000 years or more. A. versicolor, isolated most frequently from diverse ice samples in Kongsfjorden, was repeatedly isolated as well from Antarctic soils, and is also common in diverse low aw environments in temperate zones. It belongs to the most xerophilic Antarctic species, able also to grow at high levels of radioactive contamination [Onofri et al., 2004].

1.6 Eurotium

The genus Eurotium is the holomorphic ascomycete genus for Aspergillus sections Aspergillus and Restricti. Species in this genus are particularly xero- and halophilic (Pitt and Hocking, 1985a, 1985b). Its representatives have been reported to live in concentrated salt or sugar solutions at aw as low as 0.7 (Wheeler and Hocking, 1993; Martin et al., 1998). Different species of the teleomorphic genera Eurotium were often isolated from natural hypersaline water environments such as Dead Sea water and hypersaline waters of salterns around the globe. Eurotium amstelodami was isolated most frequently, followed by E. herbariorum and E. repens, E. rubrum and E. chevalieri were isolated with lower frequency [Butinar et al., 2005c].

In Kongsfjorden Eurotium species were rarely isolated, although more frequently than Aspergillus spp. They were primarily derived from glacial and sea ice. The most frequent isolate was E. repens, followed by E. amstelodami and E. rubrum. E. repens and E. rubrum were previously isolated from 8,150 years old ice and Antarctic soil [Onofri et al., 2004].

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